Method and apparatus for the non-invasive measurement of tissue function and metabolism by determination of steady-state fluorescence anisotropy

ABSTRACT

A non-invasive measurement of biological tissue reveals information about the function of that tissue. Polarized light is directed onto the tissue, stimulating the emission of fluorescence, due to one or more endogenous fluorophors in the tissue. Fluorescence anisotropy is then calculated. Such measurements of fluorescence anisotropy are then used to assess the functional status of the tissue, and to identify the existence and severity of disease states. Such assessment can be made by comparing a fluorescence anisotropy profile with a known profile of a control.

CROSS-REFERENCE TO PRIOR APPLICATION

Priority is claimed from U.S. Provisional Patent Application Ser. No.60/744,831, filed Apr. 13, 2006, the entire disclosure of which isincorporated by reference herein.

BACKGROUND OF THE INVENTION

This invention relates to the field of non-invasive measurements offunctions of biological tissues.

Structural damage in living tissues is always accompanied by functionaldeficit. However, the converse is not necessarily true. That is,functional deficit may precede irreversible structural damage for manyyears, in numerous disease states, and may therefore serve as adiagnostic indicator of early disease and a prognostic indicator ofdisease progression. For this reason, increasing attention has focusedon functional imaging of tissues, in situ, in humans.

PET scanning provides striking images of functional change; however, itsresolution is crude and its implementation expensive.

To provide high-resolution imaging of the functional state or metabolicrates of tissues non-invasively and in situ, optical techniques must beemployed. The present invention provides such an optical technique.

The functional status of bodily tissues is stoichiometrically related totissue metabolism through the well-established mechanism of respiratorycontrol. In particular, in the process of electron transfer fromsubstrates such as glucose and pyruvate to molecular oxygen, theoxidation of the flavin adenine dinucleotide FADH₂ to FAD produces 2molecules of adenosine triphosphate (ATP), while the oxidation of thenicotinamide adenine dinucleotide NADH to NAD yields 3 ATPs. ATP in turnis used to power processes within living cells that support functionwithin all living cells. Therefore, the conversions of these twonucleotides may be used to monitor cellular function and serve assensitive indicators of cellular and tissue health.

Both flavin and nicotinamide dinucleotides possess fluorescenceproperties and lifetimes that change with cellular function and both areendogenous fluorophors that are found within mitochondria as well as inenzymes within other cellular compartments in tissues. Priormeasurements of the fluorescence intensity of either or both, or of theratio of fluorescence intensities of these molecules, have been used inresearch studies by Chance and his colleagues to explore metabolism in aresearch setting. However, these approaches have never successfully beenextended to the clinical setting because they require calibration of thesystem by bringing the tissue to a uniform oxygen partial pressure of 0mm Hg by breathing an animal on 100% nitrogen. This obviously cannot bedone in humans without causing irreversible cell death.

The need to calibrate fluorescence intensity measurements arises fromthe photobleaching of fluorophors during exposure to excitation light.Moreover, fluorescence intensity is influenced by other factors thathave no relation to metabolism, such as absorption of excitation andemission light by intervening tissues. To overcome these deficiencies,some have tried to take the ratio of emissions from NADH and FAD, butthe results have not been satisfactory because the rates ofphotobleaching of these molecules are different, thus precluding aclinically useful tool.

In theory, the problems associated with fluorescence intensitymeasurements may be overcome by measuring fluorescence lifetime, namelythe decay constant of fluorescence emission following pulse excitation.However, the fluorescence lifetimes of interest would require the use offemtosecond laser pulses. Even with the use of photon countingphotomultipliers, such a technique would require, for adequatesignal-to-noise ratio, an excitation energy that would destroy tissue.

U.S. Pat. No. 5,626,134, the disclosure of which is incorporated byreference herein, describes a novel procedure for the steady-statemeasurement of fluorescence lifetime, based upon the measurement offluorescence anisotropy. The disclosed general methodology overcomes thedeficiencies of time-resolved measurements of fluorescence lifetime inboth in vivo and in vitro applications.

The present invention provides a modified technique which can be appliedto the non-invasive measurement of the steady-state fluorescenceanisotropies of flavin and nicotinamide dinucleotides within bodilytissues. As will be shown in detail below, the present invention usesmeasurements of steady-state fluorescence anisotropy to reveal tissuefunctional and metabolic status, and changes in function and metabolismthat accompany disease states. Moreover, and of singular importance,steady-state fluorescence anisotropy is employed in the presentinvention to reveal numerous aspects of metabolically-induced changes inthese endogenous nucleotides, namely, fluorescence lifetime changes thatoccur during function and metabolic change as well as conformationalchanges and changes from the unbound to bound form of these nucleotidesthat also take place during metabolism and function within bodilytissues.

In the present invention, the application of steady-state fluorescenceanisotropy measurement is a more encompassing and broader methodologyfor probing the functional and metabolic state of a bodily tissue insitu by non-invasive methods in health and disease. The method andapparatus thereby provide the first safe, sensitive, calibration-freeoptical methodology for the non-invasive measurement of metabolic rateand functional status of tissues in 2- and 3-dimensional space.

Ideally, a noninvasive optical approach that reveals function by imagingmetabolic changes should yield signals that are quantitatively traceableto function based upon the stoichiometric relationship between functionand metabolism that is imposed by respiratory control. Redox fluorometryis one well-established approach that fulfills this criterion. For thisreason, redox fluorometry employing the intrinsic fluorescence ofreduced pyridine nucleotides and oxidized flavoproteins has long beenemployed to assess cellular energy metabolism. However, suchintensity-based methods are severely limited due to photobleaching,inner filter effects and the difficulties associated with isolatingcontributions from these metabolically relevant fluorophores from thebackground autofluorescence of other endogenous fluorophores. Innerfilter effects are present in all tissues. Although measurements offluorescence lifetime can, to some extent, overcome the limitations ofintensity-based measurements, the information gleaned from suchmeasurements alone is limited, and ultra fast lifetimes require the useof high energy laser pulses that may be damaging to fragile tissues.

The present invention includes a novel method of steady-stateflavoprotein fluorescence anisotropy imaging (metabolic mapping) thatovercomes the deficiencies of intensity- or lifetime-based imaging whileretaining the essential quantitative coupling of metabolism to function.The steady-state fluorescence anisotropy (A) of a distinct molecularspecies undergoing isotropic rotational diffusion is related to theexcited state lifetime τ and the rotational correlation time φ by thefollowing equation:

$\begin{matrix}{\frac{A_{o}}{\;\overset{\_}{A}} = {{1 + \frac{\tau}{\phi}} = {1 + \sigma}}} & (1)\end{matrix}$where A_(o) is a limiting value (in the absence of rotation) given bythe relative orientation of the absorption and emission dipoletransition moments, and σ is the ratio τ/φ.

From this equation it follows that fluorescence anisotropy is aparameter with the capability of revealing changes in both orientationdistribution and excited state lifetimes with great sensitivity. Suchfunction-induced metabolic changes could arise from restrictions todiffusional motion, complex formation and molecular proximity manifestedby hetero- or homo-energy transfer. Moreover, fluorescence anisotropyand lifetime are intrinsic parameters, unlike the intensity signals usedto compute them, and are therefore insensitive to light path andgeometry. When measurements are restricted to a single intrinsicfluorophore, the effects of photobleaching are also eliminated. All ofthese advantages contribute to the capability of making reliablecomparisons over time in the same tissue in situ and between differentliving tissues.

Fluorescence anisotropy is independent of fluorophore concentration, andtherefore, unlike fluorescence intensity-based imaging or structuraltechnologies, its sensitivity is independent of the thickness of thetissue being probed. Of especial importance is the large safety marginof the method of the present invention that allows it to be employed toprobe cellular bioenergetics in living tissues in humans and the abilityto bandpass and notch filter anisotropy values provides the opportunityto reject contributions from other endogenous fluorophores.

Flavoprotein (FP) fluorescence can be excited by longer wavelength,lower energy light, is more resistant to photobleaching than pyridinenucleotides (NADH or NADPH) and is almost singularly associated withmitochondria (Koke et al, “Sensitivity of flavoprotein fluorescence tooxidative state in single isolated heart cells”, Cytobios (1981), vol.32, p. 139-145; Scholz et al, “Flavin and pyridine nucleotideoxidation-reduction changes in perfused rat liver”, J. Biol. Chem.(1969), vol. 244, p. 2317-2324). By restricting measurements to FPsteady-state fluorescence anisotropy, the risk of damage to tissues byhigh energy laser pulses is obviated by the use of lower energy lightdistributed over durations that are long relative to the excited statelifetimes. Of the numerous enzymes endowed with flavin cofactors, it hasbeen previously shown that lipoamide dehydrogenase (LipDH) dominates thefluorescence signal with lesser contributions from electron transferflavoprotein (ETF). LipDH serves as a direct probe of cellularmetabolism and function because its FAD cofactor is in directequilibrium with the mitochondrial NAD+/NADH pool, while the redox stateof ETF is indirectly affected by the NAD+/NADH ratio withinmitochondria.

It should also be noted that time-resolved fluorescence anisotropymeasurement requires the use of ultra-fast laser pulses that wouldsimilarly be destructive to tissues. For this reason, steady-statefluorescence anisotropy determination is employed in the currentinvention. Furthermore the measurement of flavin dinucleotidefluorescence anisotropy is a preferred embodiment of the technologybecause longer excitation wavelengths and thus lower excitation energiesmay be employed, thereby ensuring the safety of its use in delicatetissues such as in the non-invasive imaging of the functional status ofthe human retina, in situ, in the eye.

Although, the methodology disclosed herein is directed towardnon-invasive measurement of the fluorescence anisotropies of flavin andnicotinamide dinucleotides, and thereby the metabolic and functionalstatus of tissues, it will be apparent to one skilled in the art thatthis is a general methodology that may be applied to all endogenousfluorophors whose steady-state fluorescence anisotropies may beinfluenced by disease processes.

The present invention also includes a method for extending thesensitivity of the above-described non-invasive method by measuring thesteady-state fluorescence anisotropy of the tissue first in the restingstate and subsequently in the stimulated state. Steady-statefluorescence anisotropy maps are acquired in these two states in 2- or3-dimensional space and the anisotropy map in the resting state issubtracted point-by-point from that obtained in the stimulated state.The resultant fluorescence anisotropy map therefore reveals the capacityof the tissue to respond to stimulation. As such, the methodology hasclear application to detection and prognosis of disease states whereinthe magnitude of change to stimulation may be reduced and the spatialloci of functional and metabolic deficits identified.

SUMMARY OF THE INVENTION

The present invention provides apparatus and methods for thenon-invasive determination of the functional and metabolic status oftissues by the measurement of the steady-state fluorescence anisotropiesof one or more endogenous fluorophors, in situ, such as flavin andnicotinamide dinucleotides. By use of the present invention, therotational and vibrational motions of the isoalloxazine ring of FAD havebeen exploited to extract, by relatively simple means, a measurement offluorescence lifetime, thereby providing the first safe,high-resolution, calibration-free measurement of metabolic rate andfunction in tissues. In addition, steady-state fluorescence anisotropycan reveal conformational or binding changes in endogenous fluorophorssuch as FAD and NADH that accompany changes in cellular and tissuemetabolism.

The non-invasive measurement of functional and metabolic state of bodilytissues is accomplished by irradiating the tissue with continuouspolarized excitation light, characterizing the polarization propertiesof the emitted fluorescence from endogenous fluorophors in a manner thatprovides information sufficient to calculate steady-state fluorescenceanisotropy, and employing the calculated fluorescence anisotropy valuesto describe tissue metabolism and functional activity of the tissue.

Fluorescence anisotropy may be measured at a plurality of points inspace, thereby providing a topographic measurement of the functional andmetabolic state of the tissue. Similarly, optical serial sectioningtechniques may be employed non-invasively to provide spatial maps offunctional and metabolic states of tissues, at various depths within thetissue. Consequently, the present invention provides a sensitive,calibration-free, high-resolution methodology for the non-invasivemeasurement of the functional and metabolic state of bodily tissues inspace, time and depth.

The invention also includes a novel method of filtering the range offluorescence anisotropy values to isolate steady-state fluorescenceanisotropy measurements to a specific endogenous fluorophor.

The invention also includes an apparatus and method for measuringsteady-state fluorescence anisotropy tomographically, in 3-dimensionalspace, and in which the apparatus is coupled to a confocal scanningsystem.

The present invention also includes means for extending the sensitivityof the above-described method in disease detection. By first measuringsteady-state fluorescence anisotropy in the resting state, and thenperforming the same measurement in the stimulated state, at a pluralityof points in space, and subtracting the measurements in the restingstate from those in the stimulated state, one can detect disease-inducedreductions in the magnitude of change to stimulation, and can localizethese disease-induced deficits in 2- and 3-dimensional space.

The present invention therefore has the primary object of providing anapparatus and method for the noninvasive determination of the metabolicand functional status of biologic tissues, in situ, by measurement ofsteady-state fluorescence anisotropies of flavin adenine andnicotinamide adenine dinucleotides.

The invention has the further object of providing an apparatus andmethod for the non-invasive determination of the metabolic andfunctional status of biologic tissues in situ topographically, in2-dimensional space, and tomographically in 3-dimensional space.

The invention has the further object of enabling non-invasive earlydetection of disease by measuring the effects of the disease on thefunctional and metabolic state of tissues.

The invention has the further object of enabling prognosis of diseaseprogression by virtue of the change in functional and metabolic state oftissues over time, in the same patient, and the comparison of apatient's changing metabolic profiles to a normative database of diseaseprogression established in clinical trials.

The invention has the further object of enabling detection of diseaseprior to irreversible structural damage, by measurement of functionaland metabolic changes that precede such irreversible structural damage.

The invention has the further object of guiding therapeuticinterventions that are directed toward ameliorating disease, bynon-invasive and non-destructive monitoring of the effects oftherapeutic interventions on the metabolic and functional status oftissues.

The invention has the further object of determining the specificlocations of disease-induced deficits in tissue function, by measuringsuch tissue in a resting state and in a stimulated state, and bycomparing such measurements precisely at various points in a two- orthree-dimensional space.

The reader skilled in the art will recognize other objects andadvantages of the invention, from a reading of the following briefdescription of the drawings, the detailed description of the invention,and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic diagram of an imaging apparatus used tomeasure tissue function and metabolism, according to the presentinvention.

FIG. 2 provides a schematic diagram of an imaging apparatus, madeaccording to the present invention, for deriving three-dimensional mapsof tissues.

FIGS. 3A-3F provide diagrams illustrating the use of the presentinvention to detect changes in the condition of eye tissues in an animalby measurement of fluorescence anisotropy, where FIG. 3A provides afrequency histogram showing the variation of fluorescence anisotropyunder different conditions, FIGS. 3B and 3C provide grayscale maps offluorescence anisotropy values in the eye of an animal under differentconditions, FIG. 3D provides infrared scans of treated and untreatedeyes of an animal, and FIGS. 3E and 3F provide angiograms depicting thecondition of the eye of an animal.

FIGS. 4A-4F provide diagrams illustrating the use of the presentinvention to detect diabetic retinopathy, where FIGS. 4A-4D providefrequency histograms showing the variation in fluorescence anisotropyunder various conditions, FIG. 4E provides a graph summarizing theresults, and FIG. 4F provides a scan of the retina showing the area ofinterest.

FIGS. 5A-5H illustrate the use of the present invention to analyzetissue function by comparing fluorescence anisotropy maps taken in aresting state and in a stimulated state, wherein FIGS. 5A-5C depict aportion of the retina in resting and stimulated conditions and showing acomparison of the two states, FIG. 5D provides a scan of the region ofinterest, and FIGS. 5E-5H provide graphs analyzing the results.

FIGS. 6A-6G illustrate the use of the present invention in detectingglaucoma, where FIG. 6A-6B provide grayscale maps of fluorescenceanisotropy values in patients with and without glaucoma, FIG. 6Cprovides a graph of anisotropy values for various patients, FIGS. 6D-6Eprovide diagrams showing fluorescence anisotropy values for patientswith and without glaucoma, and FIGS. 6F-6G provide diagrams showingcorresponding results, for the same patients, using conventionaldiagnostic techniques.

FIGS. 7A-7F illustrate the use of the present invention in detectingocular hypertension, where FIGS. 7A and 7D provide frequency histogramsof fluorescence anisotropy for various conditions, and FIGS. 7B, 7C, 7E,and 7F provide tomographs showing the eye under various conditions.

FIGS. 8A and 8B illustrate the use of the present invention to evaluatethe effectiveness of treatment for diabetic retinopathy, where FIG. 8Aprovides a two-dimensional map of fluorescence anisotropy of a region ofthe retina, showing treated and untreated areas, and FIG. 8B provides agraph showing fluorescence anisotropy values measured along the linedrawn in FIG. 8A.

FIG. 9 provides a graph showing fluorescence anisotropy values generatedfrom measurements of a human retina, according to the present invention,the figure showing a curve based on measurements taken while a subjectis breathing pure oxygen, and a curve based on measurements taken whilethe subject is breathing pure air.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 provides a schematic diagram of an apparatus 10 designed for thetopographic, 2-dimensional, mapping of tissue function and metabolismnon-invasively in an imaged tissue. Specimen 12 includes one or morebiological tissues which contain one or more substances, calledfluorophors, which fluoresce when illuminated.

Specimen 12 is illuminated with nonpolarized visible light by afiberoptic illuminator 14 utilizing a tungsten source 16. The radiantenergy of a xenon arc lamp 18 is gathered by a collector lens 20, and isselectively allowed to pass through shutter 22. The light then passesthrough a Glan-Thompson polarizer 24 (obtained from Ealing, Inc.).

The light is spectrally shaped by an excitation filter 26 and is thenreflected by a dichroic mirror 28 (obtained from Omega Optical) whichreflects excitation wavelengths through an objective lens 30 to theimaged tissue, causing fluorophors in the tissue to fluoresce.

Emitted luminescence from the excited tissue 12 is gathered by theobjective lens 30 and passes through the dichroic mirror 28, and throughan emission filter 32, which passes emission wavelengths to a Wollastonprism polarizer 34 which resolves the emitted fluorescence into itslinearly polarized components parallel 36 and perpendicular 38 to theplane of excitation polarization. The parallel and perpendicular vectorcomponents 36, 38 are respectively and simultaneously detected by theCCD (charge coupled devices) chips of two video cameras 40A and 40B(e.g. Xybion model 250).

Alternative optical detectors with sufficient spatial resolution, suchas slow scan chilled CCD cameras, SIT or ISIT tube cameras, orphotodiode arrays (not shown), would also be suitable for the detectionof the two-dimensional distributions of the parallel and perpendicularcomponents of the emitted fluorescence.

The outputs of the two video cameras are digitized by two digitizingboards 42A and 42B (such as sold by Imaging Technologies or under thedesignation model DT3851 by Data Translation) within a microcomputer 44,(e.g., an IBM or equivalent computer, preferably having a processingchip operating at 33 or 66 MHz). Such a device is sufficient for thetask and for subsequent image processing prior to display on the monitor46.

FIG. 2 provides a schematic of an apparatus designed for the tomographic(3-dimensional) mapping of tissue metabolism and function, according tothe present invention. Light from one tunable argon laser (e.g. MellesGriot part number 35 LAP 321-240) and two diode lasers (e.g. MellesGriot part number 56 ICS 008-HS) input light into the confocal device bymeans of a trifurcated single mode polarization fiber (such as sold byEaling Inc. under the designation of catalog number 34-5223), denoted inFIG. 2 as SMPPF.

The excitation or imaging wavelength is selected by computer-controlledselection of the appropriate laser, and in the case of the argon laserby tuning the laser and by the additional use of computer-selectablenarrow bandpass filters. The argon laser permits excitation at 488 nmfor fluorescence anisotropy measurements from FAD, as well as 514 nm forobserving the tissue in reflection mode. The diode lasers permitobservation of the tissue at 730 nm or 830 nm in reflection mode. Otherlight sources emitting light at other wavelengths may be employed inthis embodiment.

The small diameter fiber (<150 μm) serves as a point source that iscollimated by a lens system (L1 and L2). The collimated light passesthrough a linear polarizer P1 (which may be obtained from MeadowlarkOptics, part number C001298) and then passes through a custom fabricatedLCD twisted nematic switch (such as Meadowlark Optics part no. C001700).The twisted nematic switch allows rapid rotation of the plane ofpolarization of the collimated light incident on it by 90° with risetimes of the order of 20 msec. The linearly polarized light at eitherorthogonal plane of polarization is reflected by a front surface mirrorand the collimated polarized light passes through a dichroic mirror,designated DCM in FIG. 2 (such as can be obtained from Chroma or OmegaOptics) to fall incident on two servo-controlled mirrors VSM and HSMthat serve as a scanning system.

Movement of the mirrors VSM and HSM causes light to scan a tissue samplein a raster fashion, in vertical and horizontal directions,respectively, thereby performing a two dimensional scan of the tissue.

The light for either fluorescence excitation, or imaging in reflectionmode, at any of three wavelengths, passes through the first component ofthe objective, lens L3, and subsequently through the second component ofthe objective, housed in the outer shell, L4. Fluorescence emission orreflected light from the tissue returns through objective lenses L4 andL3 and is scanned by mirrors VSM and HSM onto the dichroic mirror DCM.The dichroic mirror DCM reflects light at wavelengths >500 nm, and thislight, in turn, passes through a longpass interference filter (such asChroma part number HQ5101p) that transmits light >500 nm and rejectslight <500 nm by 5 orders of magnitude (O.D. >5 from 300-500 nm).

Light of wavelengths >500 nm is imaged onto a pinhole by L5 and thenpasses through a linear polarizer P2 (which may be Meadowlark Optics,part number C001298) with its plane of polarization set parallel to thatof P1. Light passing through P2 falls onto the detector surface of acustom-made high-sensitivity photomultiplier module (such as Hamamatsupart number H9656-20MOD W/2.5 MHz AMP), depicted as a “detector” in FIG.2. The output of the photomultiplier module is digitized and bitmapimages of 512×512 pixels are saved in random-access memory (RAM) andstored to disk. Other detectors such as avalanche photodiodes mayreplace the photomultiplier. The resolution of the scanning may beadjusted to any desired resolution by selection of alternative scanningsystems and servo controllers.

For fluorescence anisotropy imaging, rotation of the plane ofpolarization by the twisted nematic switch is synchronized with the fullframe acquisition speed, thereby exciting the tissue at orthogonalplanes of polarization in succession. In the embodiment shown, the scantime for a full frame is 28 msec. By actuating the twisted nematic,fluorescence emission is resolved into vector components parallel andperpendicular to the plane of polarization of P2, thus providing vectorcomponents I_(∥) and I_(⊥) employed in the calculation of fluorescenceanisotropy, according to the following equation:

$A = \frac{I_{\parallel} - {GI}_{\bot}}{I_{\parallel} + {2{GI}_{\bot}}}$where G is an empirical correction factor used to correct fortransmission efficiency in parallel and perpendicular planes.

The system is automated and synchronized to acquire sequentialfluorescence emission images in parallel and perpendicular planes atspeeds ranging from 6 to 25 Hz. Any desired number of framescorresponding to orthogonal planes of polarization may be acquired andaveraged to eliminate shot noise, and the images in the case of tissuesthat are moving, such as in the eye, are aligned by software fortranslational and rotational movements.

The tissue is brought into focus by moving the outer shell with theinner assembly fixed in position. The entire inner assembly of theconfocal metabolic mapper is motorized for translation in the xdirection as depicted and the inner assembly may be moved in selectablediscrete steps by a stepping motor.

Sectioning the tissue at desired resolution within the depth of thetissue for both fluorescence anisotropy maps and reflection images isperformed by appropriate selection of the size of the pinhole and thesize of the displacement of the inner assembly by the stepping motor. Inthis manner, tomographic fluorescence anisotropy maps in 3-dimensionalspace may be acquired. Similarly for fluorescence anisotropy mapping ofother endogenous fluorophors such as NADH, alternative pairs of DCM andblocking filters may be switched in by computer-actuated linearsolenoids.

To implement a system that has appropriate sensitivity to metabolic andfunctional changes in the examined tissue, it becomes necessary torestrict steady-state anisotropy to the relevant fluorophor thatprovides such information, e.g., FAD. In other words, all tissuescontain a number of endogenous substances that fluoresce at a givenexcitation wavelength, and it becomes necessary to extract informationfrom the metabolically-relevant fluorophor from the total backgroundfluorescence arising from multiple fluorophors within the tissue.

In principle, one can extract information from the fluorophor ofinterest by spectrally shaping excitation and emission filters. However,this procedure is rarely sufficient to accomplish this important task.For this reason, the present invention provides a new approach,described below, that deals with the range of steady-state fluorescenceanisotropy values calculated from fluorescence emissions.

To develop a means for isolating fluorescence anisotropy to a givenfluorophor, the tissue can be imaged using the optical device shown inFIG. 1, wherein the CCD detectors for parallel and perpendicular vectorcomponents are replaced by fiber optic coupled high sensitivity gratingspectrometers (e.g. Ocean Optics, part number USB4000-VIS-NIR) thataverage over the full field of view. In this manner, fluorescenceanisotropy arising from the tissue can be determined as a function ofwavelength. Using the formula given above (and in the aforementionedU.S. Pat. No. 5,626,134), one can produce software which will calculatefluorescence anisotropy from vector components of fluorescence emissionparallel and perpendicular to the plane of polarization of theexcitation light, and to display fluorescence anisotropy as a functionof emission wavelength.

By appropriate spectral shaping of the incident excitation light andbandpass filtering, i.e., limiting the range of collected anisotropyvalues, the emission anisotropy spectrum can be constrained closely tomatch published values of the fluorescence emission spectrum of knownfluorophors, such as FAD or NADH. The same procedure can be applied toisolate contributions from other endogenous fluorophors, the changes influorescence anisotropy of which may be relevant to other diseaseconditions. The results of such a procedure are summarized in FIG. 9,discussed below.

To test the ability of the above-described procedure to isolatesteady-state fluorescence anisotropy due to FAD only, a human retina wasimaged with 488 nm excitation light and the fluorescence anisotropycalculated from the emitted fluorescence vector components determined asa function of wavelength. The measurements were performed with thesubject breathing either room air or 100% oxygen. Fluorescenceanisotropy values were bandpass filtered until fluorescence anisotropyplotted as a function of wavelength closely matched the fluorescenceemission spectrum of FAD as found in the literature. Since fluorescenceanisotropy changes in magnitude when the subject breathes oxygen, ascompared to room air, it is possible to test the efficacy of this novelprocedure by normalizing the results obtained under room air and 100%oxygen conditions. If the procedure of the present invention iseffective, then the two graphs should overlap when normalized. This isprecisely what is obtained in practice. FIG. 9 provides a graph showingcurves representing the two conditions of measurement.

In summary, the present invention enables the user to fit an anisotropyprofile to a profile of a known fluorophor, by appropriate adjustment offilter parameters. Such adjustment would preferably, but notnecessarily, be done entirely by software. Then, a device made accordingto the present invention could be directed at an unknown sample, withthe parameters set as previously determined, and one would know, withconfidence, that the results obtained were due to the particularsubstance of interest.

More specifically, in the example discussed above, in which thefluorophor of interest is FAD, one would select the parameters so thatthe anisotropy diagram fits the known profile for FAD, meaning that thesystem has isolated the contribution from FAD from the contributionsfrom all other fluorophors in the sample. Subsequent operation of themachine, on an unknown sample, would then yield results based only onthe effects of FAD, and not on the other fluorophors in the sample.Thus, the present invention provides a reliable means of detectingfluorescence anisotropy from a particular fluorophor, even when thesample contains multiple fluorophors, and even when the contributionsfrom the other fluorophors might otherwise mask the effect of thefluorophor of interest.

Moreover, a novel adaptation of the general methodology allows thepreferred embodiment to provide greater sensitivity in non-invasivelydetecting and localizing disease-induced reductions in function andmetabolic capacity within tissues. This may be accomplished in the humanretina, in situ, by the following paradigm in which the retina is firstimaged for orientation with 830 nm light, supplied by Laser 3 of FIG. 2,to which the retina is unresponsive, and then obtaining steady-statemetabolic maps of the retina within 100 msec., which falls well withinthe latency of the metabolic response of the retina to saturating light.This procedure provides a steady-state fluorescence anisotropy map ofthe retina in the dark, resting state. Subsequently, the retina isimaged at 830 nm followed by 20-30 sec of flickered blue (488 nm) orgreen (514 nm) light supplied by the argon laser, Laser 1, at flickerrates ranging from 6-13 Hz and fluorescence anisotropy images acquiredduring flicker, thereby providing a measurement of the light, stimulatedstate of the retina. Dark and light fluorescence anisotropy maps can bealigned and subtracted pixel-by-pixel to yield a 2- or 3-dimensional mapof the functional capacity of the retina to respond to lightstimulation. In addition, by selection of different types of spatial,spectral or temporal configurations of light stimulation, one canisolate function and metabolism to different cell layers and typeswithin the retina.

Although the preferred embodiment employs light stimulation in the caseof the retina, it should be apparent that the same preferred embodimentmight be applied to other bodily tissues by selecting some other mode ofstimulation appropriate to the tissue being examined.

FIGS. 3A-3F illustrate the use of the present invention to detectchanges in tissue metabolism. In the experiment represented by thesefigures, one eye of a monkey was subjected to slight damage bylaser-induced occlusion of a large vein. The latter condition is knownin the art as large branch vein occlusion (BRVO). FIGS. 3A-3F illustratethe ability of the present invention to detect this damage.

In the experiment represented by FIGS. 3A-3F, fluorescence anisotropy ofthe macula was measured under the following three conditions. First,fluorescence anisotropy of the macula of the damaged eye was measured,over a 20° visual field centered at the macula, while the animal wasbreathing pure oxygen. Secondly, fluorescence anisotropy of the maculaof the undamaged (“contralateral”) eye was measured, also while theanimal was breathing pure oxygen. Thirdly, fluorescence anisotropy ofthe macula of the undamaged eye was measured while the animal wasbreathing room air. As shown in FIG. 3A, all three of these conditionsyielded distinct graphs.

The normal, untreated eye shows increased fluorescence anisotropy valueswhen the animal breathes pure oxygen as compared to room air. Also, thedamaged eye provides significantly lower values of fluorescenceanisotropy when the animal breathes pure oxygen, as compared to theuntreated eye when the animal breathes room air. All comparisons weremade with the same animal.

FIG. 3A therefore shows the use of the present invention in detectingsubtle metabolic changes, insofar as the tissue metabolism is enhancedwhen the animal breathes pure oxygen, and is depressed by reducing bloodflow to the tissue due to the induced vein occlusion. This induceddamage would not have been detectable using conventional methods.

FIG. 3B provides a grayscale spatial map of fluorescence anisotropyvalues in the damaged eye, while the animal was breathing pure oxygen,over a 20° visual field. The dark areas correspond to high values andlight areas to low values. FIG. 3C provides a similar map for theundamaged eye, also while the animal was breathing pure oxygen. Notethat the fluorescence anisotropy values represented in FIG. 3B, whiledepressed across the entire 20° field as compared to FIG. 3C, show themost pronounced depression in the macula. Conversely, FIG. 3C showsincreased anisotropy values across the entire field, as compared to FIG.3B, with the most pronounced increase occurring at the macula.

FIG. 3D provides near-infrared scans, respectively, of the damaged andundamaged eye of the animal. FIG. 3E provides a fluorescein angiogramshowing the site of the laser-induced branch vein occlusion. FIG. 3Fprovides a fluorescein angiogram of the damaged eye, centered at themacula, showing the tortuosity of vessels encroaching the macula.

The comparison of the curves in FIG. 3A can be performed visually, by ahuman operator, or the comparison could be automated, and performed by aprogrammed computer. In this regard, the computer shown in FIG. 2 shouldbe considered a means for performing such comparison.

In grayscale metabolic maps, the dark areas correspond to high valuesand light areas to low values. The relationship between grayscale andquantitative values of fluorescence anisotropy is enhanced linearly tothe same extent in images within the figure. Note that in this and allfollowing figures, the frequency histograms are a plot of the frequencyof occurrence of different fluorescence anisotropy values within themeasurement area.

FIGS. 4A-4F illustrate the use of the present invention to identifydisease. In particular, these figures illustrate the use of steady-statemeasurements of fluorescence anisotropy to identify diabeticretinopathy, in human retinal tissue in which the disease is accompaniedby reduced metabolism of the retina. The invention is used to identifyboth moderate, nonproliferative diabetic retinopathy and mildproliferative diabetic retinopathy (PDR) in the temporal retina.

FIG. 4A provides a frequency histogram of fluorescence anisotropy valuesfor a patient with moderate nonproliferative diabetic retinopathy, withthe patient breathing room air. FIG. 4B provides a frequency histogramof fluorescence anisotropy values in normal, age and gender-matchedcontrol subjects, also breathing room air. Note that the mean of thehistogram in FIG. 4A is shifted to lower values, and is wider, comparedto the measurements taken for normal subjects, in FIG. 4B.

FIG. 4C provides a frequency histogram of fluorescence anisotropy valuesin the same patient represented in FIG. 4A, with the patient breathingpure oxygen. Note that the mean of anisotropy values for this patient isapproximately equal to that of the control subject breathing room air(FIG. 4B).

FIG. 4D provides a frequency histogram of fluorescence anisotropy valuesof an age and gender-matched control subject breathing pure oxygen. Notethat the histogram of FIG. 4C is wider than that of the control subjectbreathing pure oxygen (FIG. 4D).

FIG. 4E provides a graph showing normalized mean values of patients withmild PDR compared to normal age- and gender-matched controls (n=20)error bars are +/−1 S.E.M. The bars labeled with asterisks indicate thatthe results had statistical significance at the P value shown. Themiddle bar represents a test on a single patient, too small of a samplefor statistical significance. Nevertheless, the mean fluorescenceanisotropy value of that patient, who had diabetes for about ten years,without overt retinopathy, shows intermediate depression of fluorescenceanisotropy values, relative to the first and third bars, as demonstratedby fluorescein angiography.

FIG. 4F provides a near infrared scan of a retina, showing the area ofinterest in the temporal retina from which fluorescence anisotropyfrequency histograms were generated.

As before, the computer shown in FIG. 2 can be considered a means forperforming the comparisons among the curves shown in FIGS. 4A-4D, and/oramong the bars in FIG. 4E.

FIGS. 5A-5H illustrate the use of the present invention in detecting andlocalizing functional and metabolic deficits in tissues byquantitatively subtracting the fluorescence anisotropy maps of a tissuein the resting state from those obtained in the same tissue in astimulated state. The invention thereby allows quantitativedetermination and localization of regions within the tissue that showreduced capacity to respond to stimulation.

FIG. 5A provides a metabolic map of a 20° field, centered at the opticdisc of the retina, showing fluorescence anisotropy in space, in thedark, resting state. Note that, in the dark, the temporal retinal andtemporal neuroretinal rim are depressed relative to the nasal retina andneuroretinal rim.

FIG. 5B provides a metabolic map of the same region as FIG. 5A, after 20seconds of flickered light stimulation at 12 Hz. Note that flickeredlight causes fluorescence anisotropy values to increase across theentire field relative to FIG. 5A.

FIG. 5C shows the result when the images of FIGS. 5A and 5B werealigned, and one image was subtracted from the other, pixel-by-pixel, toyield a functional map of the 20° visual field (b-a) in response toflickered light stimulation. Note that the temporal retina and temporalneuroretinal rim become dominant. The relationship between grayscale andquantitative values of fluorescence is enhanced linearly to the sameextent in all images within the figure and the lighter regionscorrespond to high values and the darker to low.

FIG. 5D provides an infrared scan of the same region shown in FIGS.5A-5C, for orientation.

FIG. 5E provides a graph showing the vertical average between thehorizontal lines in FIG. 5A (darkness) as a function of distance inpixels. The smooth curve is a 20th order Chebyshev polynomial fit(r²=0.9818). The values are plotted from the temporal to nasal sides.The residuals were normally distributed.

FIG. 5F provides a graph of the vertical average between the horizontallines in FIG. 5B (after flickered light) as a function of distance inpixels. The smooth curve is a 20th order Chebyshev polynomial fit(r²=0.7806). The values are plotted from the temporal to nasal sides.The residuals were normally distributed.

FIGS. 5G and 5H provide frequency histograms of fluorescence anisotropyvalues for the 20° field and disc, respectively, in darkness and afterflickered light.

The computer and monitor shown in FIG. 2 can be used as means forperforming the above analyses. The patterns shown in FIGS. 5A-5D can bedisplayed on the monitor, while the calculations needed to form thedifference image of FIG. 5C can be performed by the computer. Thecomputer may also be used to generate the fitted curves of FIGS. 5E and5F, and to generate and compare the curves shown in FIGS. 5G and 5H.

FIGS. 6A-6G illustrate the use of the present invention to identifydisease states non-invasively in humans. In this example, the disease isprimary open angle glaucoma (POAG).

FIG. 6A provides a grayscale map of steady-state fluorescence anisotropyvalues in a 20° field, centered at the optic disk, in a retina of anormal (control) patient. FIG. 6B provides a similar map for a patientwith mild POAG.

FIG. 6C provides graphs of fluorescence anisotropy values, across theoptic disk, for a normal patient, a patient with mild POAG, and apatient with severe POAG. Thus, the present invention is capable of notonly identifying the presence of the disease, but also of distinguishingamong degrees of its severity.

FIGS. 6D and 6E provide diagrams showing fluorescence anisotropy valuesmeasured at different areas of the neuroretinal rim of a normal control(FIG. 6D) and a patient with mild POAG (FIG. 6E). Note that the valuesof the patient with mild disease are depressed at all loci.

FIGS. 6F and 6G illustrate measurements made with the Heidelberg HRTII,the device most commonly used to detect glaucoma, for the normalpatient, and the patient with mild disease, of FIGS. 6A and 6B,respectively. Thus, the prior art device fails to distinguish betweenthe normal control and the patient with mild disease.

The computer, shown in FIG. 2, can therefore be programmed to generateand analyze curves such as those shown in FIG. 6C, and to producediagnoses, indicating whether a disease state exists, and the extent ofits severity.

FIGS. 7A-7F illustrate the use of the present invention to detect subtlechanges in blood flow and reduced function and metabolism of tissues ofthe eye, by measuring steady-state fluorescence anisotropy. Inparticular, these figures, taken from observations of the eyes of amonkey, illustrate the use of the present invention in detecting ocularhypertension (OHT). The present invention detects subtle effects whichare not always observable by use of optical coherence tomography, whichis the most sensitive known method of the prior art.

FIG. 7A provides frequency histograms of fluorescence anisotropy valuesfor 20° visual fields centered at the optic disks of a normal, untreatedeye and an eye having OHT, of the same monkey, under conditions wherethe monkey was breathing pure oxygen. The values of fluorescenceanisotropy for the diseased eye are lower than the values for thehealthy eye. FIG. 7B shows a normal, untreated optic disk, using ocularcoherence tomography (OCT), and FIG. 7C shows an optic disk having OHT,again using the technique of OCT. The lowered values of fluorescenceanisotropy for the diseased eye correspond to the excavation of the diskshown in FIG. 7C.

FIG. 7D provides frequency histograms of fluorescence anisotropy valuesof 20° visual fields centered at the macula, for a normal untreated eye,and an OHT eye, under conditions where the animal was breathing pureoxygen. Note the marked depression of fluorescence anisotropy values forthe ocular hypertensive macula as compared to macula of the normal eye.This pronounced depression of fluorescence anisotropy values withincreased intraocular pressure is present despite the fact that noevidence of structural damage is revealed by OCT. FIGS. 7E and 7Fprovide tomographs of a normal and diseased eye, respectively, of thesame monkey. All of the OCT maps were of very high quality due to themonkey being paralyzed, thereby removing motion artifacts normallyassociated with OCT measurements. All data in FIGS. 7A-7F came from thesame animal. Comparison of the curves shown in FIGS. 7A and 7D can beperformed by the computer shown in FIG. 2.

FIGS. 8A-8B illustrate the use of the present invention to reveal theefficacy of treatment interventions, through measurement of steady-statefluorescence anisotropy. In this case, panretinal photocoagulation wasused to treat proliferative diabetic retinopathy.

FIG. 8A depicts a fluorescence anisotropy 2-dimensional map of a regionin the peripheral retina of a diabetic patient with proliferativedisease, showing untreated tissue and two laser photocoagulated smallregions. A computer-drawn line for analysis is placed across the regionshowing untreated and treated areas. FIG. 8B provides a graph showingsteady-state fluorescence anisotropy values, measured along the lineprofile. As explained above, tissue metabolism is correlated withfluorescence anisotropy. The graph shows that tissue metabolism isincreased by laser treatment, thereby providing an indication of theefficacy of the treatment.

While the examples given above deal with disease states within the humanretina, it should be apparent to one skilled in the art that themethodology of the present invention may be used to detect changes thatfall within two general classes of biologic change, namely angiogenesisand apoptosis.

Angiogenesis is the process of development of new blood vessels to meetthe metabolic requirements of the tissue, which in normal developmentfollows a coordinated course leading to the formation of competent bloodvessels that follow a pattern governed by the changes in metabolism thatoccur during development. However, numerous disease states involveaberrant metabolism that leads to the uncoordinated florid growth of newblood vessels that may be leaky or incompetent.

One notable class of diseases to which the methodology of the presentinvention may be applied is in cancers that develop and can havepotentially lethal consequences. The sequence of events that isgenerally accepted in the field of angiogenesis is that tissue hypoxia,low levels of tissue oxygenation, resulting from increased metabolism orreduced blood flow, in turn cause upregulation of genes that expressvascular endothelial growth factor (VEGF). VEGF has, in addition toother angiogenic growth factors, been demonstrated to cause new bloodvessel growth. The example of diabetic retinopathy, described above,illustrates one such case. In cancers of numerous origin and tissueinvolvement, tumors show increased metabolism that may be detected bythe present invention before they may be identified visually. Tumors canonly grow in size if new blood vessels develop to nourish the tumor andstructural technologies employing contrast agents in CT scanning and MRIscanning look for enhancement of the visualized mass upon injection ofcontrast agents that flow through the newly formed blood vessels. Thepresent invention allows the detection of increased metabolism prior tonew blood vessel growth, allowing tumors to be ablated at their earlieststages, thereby reducing the need for extensive chemotherapeutic andradiation interventions.

Wound healing presents another interesting application of themethodology of the present invention. A key question in surgical removalof dead tissue is the distinction between living and dead (metabolicallyinactive tissue). Similarly new blood vessels need to grow to supportthe joining of two living tissues. The present invention can be used todistinguish between living and dead tissue and tissue capable ofsustaining coordinated blood vessel growth.

It should be apparent to one skilled in the art that the above is not anexhaustive list of diseases to which the methodology may be applied. Forexample, there are a host of mitochondrial diseases that are geneticallypassed from generation to generation. Such mitochondrial diseases may bedetected and imaged using the techniques of the present invention, basedupon the aberrant metabolic consequences of these diseases.

Apoptosis is an orchestrated form of cell death that proceeds normallyin the continuing renewal of tissues within the body. However, diseasestates may, as in the case of angiogenesis, cause the process to becomeuncoordinated, with severe consequences to patients. The presentinvention provides one example of apoptosis that proceeds in anaggressive manner, in the cases of glaucoma and other opticneuropathies.

Glaucoma belongs to a large family of neurodegenerative diseases thatinclude Huntington's, Parkinson's and Alzheimer's diseases. The methodsof the present invention may be applied to detect other diseases of thistype at early stages.

For example, Alzheimer's disease is associated with destruction ofretinal nerve fibers and ganglion cells. However, structural imaging inAlzheimer's disease only reveals the disease in the retina at relativelyadvanced stages when cells have died. It is well known that metabolicdysfunction precedes apoptosis in all tissues and the methodology hereindisclosed may allow the detection of Alzheimer's and otherneurodegenerative diseases at their earliest stages during a routine eyeexam.

One interesting case in which the physician intentionally inducesapoptosis, cell death, is in the area of cancer treatment by means ofchemotherapeutic agents and/or radiation. Just as cellular dysfunctionand reduced metabolism precede apoptosis, energy is required to initiatethe final step of apoptosis, causing the cells to die and pass the pointof no return. This burst of metabolism may be visualized usingsteady-state flavoprotein fluorescence anisotropy imaging, therebyallowing the oncologist to provide sufficient chemotherapeutic agents tokill cancerous cells while halting or reducing the dose of such agentsto avoid the dire side effects of chemo- and radiation-therapies.

An exceedingly important application of the methodology of the presentinvention is in the field of anesthesiology. The goal of theanesthesiologist, during all procedures, is to protect the brain fromirreversible damage. Indeed, cognitive losses have been reported in longsurgeries such as bypass surgery and carotid endarterectomies, to nametwo. The retina, in addition to being a part of the brain, is the mostmetabolically active part of the brain. Currently, all that may bemeasured is the partial pressure of oxygen in the blood while it isbrain tissue oxygenation and metabolic rate that is critical toprotecting the brain from irreversible damage. By imaging the retinawith the technology of the present invention, during surgeries, it willbe possible to measure reduced tissue metabolism in the retina thatprecedes dysfunction and cell death within the rest of the brain, andthereby to adjust oxygen supply to the patient to avoid irreversiblebrain damage. Just as too little oxygen can be damaging so may supplyingtoo much oxygen for prolonged periods of time and that is why themethodology of the present invention may, when compared to a database ofnormal metabolic levels, be used to adjust oxygen delivery to thepatient to provide appropriate and protective levels of oxygen. Thecomparison and use of a database for all disease states anddetermination of normal values will permit threshold values for normalmetabolism to be determined and allow treatment modalities to beemployed to restore regions of tissue metabolism to their normal levels.Such databases can be determined in clinical trials.

The present specification has focused thus far on one of the substratesof metabolism, namely oxygen. Glucose is, of course, the second majorsubstrate in the pathway of oxidative metabolism. Numerous methodologieshave been proposed, and some implemented, for the measurement of bloodglucose. However, just like oxygen, it is the level of glucose suppliedto the mitochondria of living cells within tissues that is mostimportant. The methods of the present invention provide a uniqueopportunity to measure mitochondrial metabolism and glucose supply whenthe second substrate—oxygen—is held constant. Indeed, in humans withdiabetes, the oxygen level supplied to the patient in everyday life isthe percentage of oxygen in room air, which remains constant. Thereforethe present invention provides an entirely different means of titratingglucose and insulin in diabetic patients while achieving the goal ofdoing so by a noninvasive method. Handheld devices used in the homehealthcare field may be developed that monitor mitochondrial functionand its change by blood glucose, insulin or oral diabetic drugs that maybe performed by flashes of polarized light to the eye. Unlike numerousother approaches that center on blood glucose where the signal-to-noiselevel levels may be low, the present methodology achieves highsignal-to-noise levels as demonstrated in the examples herein provided.

Since mitochondrial metabolism is essential to life, it will be apparentto those skilled in the art that the methodology described herein mayhave far-reaching applications to multiple medical disciplines.

Similarly, the implementations of the methods presented in theschematics of FIGS. 1 and 2 are but two of many that may be devisedbased upon the present method. Steady-state flavoprotein fluorescenceanisotropy imaging may be applied to any tissue that may be imageddirectly—the eye, skin, cervix etc.—and may easily be incorporated intoendoscopic devices used to examine internal organs such as theesophagus, stomach, gut or lungs, or laparoscopic and arthroscopicdevices that are currently in use in medicine. In addition, miniaturizedcatheters that may be inserted into blood vessels and other smallcaliber regions may be fabricated employing the method of the presentinvention, by means of the use of single mode polarization preservingoptical fibers.

The invention can be modified in other ways which will be apparent tothose skilled in the art. The specific arrangements shown in FIGS. 1 and2 can be varied considerably, and there are many ways to implement themethods described above. Such modifications should be considered withinthe spirit and scope of the following claims.

What is claimed is:
 1. A non-destructive method of evaluating thefunctional capacity of a living human tissue in situ in an intact livinghuman being, comprising: (a) non-destructively imaging a first restingsteady-state fluorescence anisotropy map of said tissue within a latencyperiod of metabolic change in response to a stimulus that changesmetabolism and function by the following steps: (i) irradiating thetissue with continuous linearly polarized light sufficient to causeendogenous lipoamide dehydrogenase (LipDH) to fluoresce within thelatency period of metabolic change to light, and capturing thefluorescence emission prior to the excitation light causing the tissueto change its metabolism; (ii) resolving the emitted fluorescence fromsaid LipDH into vector components parallel and perpendicular to theplane of the exciting polarized light; (iii) calculating thesteady-state fluorescence anisotropy (A) of said LipDH from the resolvedvector components; and (iv) constructing a steady-state fluorescenceanisotropy map image of said tissue; (b) non-destructively imaging asecond stimulated steady-state fluorescence anisotropy map of saidtissue for a second metabolic or functional state by the followingsteps: (i) irradiating the tissue with continuous linearly polarizedlight sufficient to cause endogenous LipDH to fluoresce after thelatency period of metabolic change to light, and capturing thefluorescence emission after the stimulus has caused the tissue to changeits metabolism; and (ii) repeating parts (ii), (iii) and (iv) of step(a); (c) determining the functional capacity of the tissue bysubtracting point by point or pixel by pixel the first restingsteady-state fluorescence anisotropy image from the second stimulatedsteady-state fluorescence anisotropy image, over the entire imaged fieldor within areas of interest; and (d) comparing the functional capacityof the tissue under evaluation to a database of normal metabolic levelsobtained from control tissue similarly determined by steps a-c to revealdysfunctional areas in the tissue under evaluation.
 2. The method ofclaim 1, wherein said first and second maps comprise two dimensionalsteady-state fluorescence anisotropy images.
 3. The method of claim 2,wherein said two dimensional steady-state fluorescence anisotropy imagesare obtained at a plurality of depths within the tissue.
 4. The methodof claim 1, wherein three-dimensional volume maps are constructed forboth the tissue subject to evaluation and the control tissue.
 5. Themethod of claim 4, wherein metabolic and functional deficits arerevealed in three dimensional space.
 6. The method of claim 1, whereinsaid stimulus includes light stimulation and stimulation by means otherthan light.
 7. The method of claim 5, wherein deficits resulting fromdisease or effects of a therapeutic or surgical treatment are localizedin three dimensional space when compared to three dimensional volumes offunctional capacity determined in normal untreated control subjects. 8.The method of claim 1, wherein said tissue subjected to evaluationcomprises ocular tissue.
 9. The method of claim 6, wherein the stimulusis light.
 10. The method of claim 1, wherein said LipDH fluorophoremission is isolated from metabolically inert fluorophor emissions basedon their respective steady-state fluorescence anisotropy values.
 11. Themethod of claim 1, wherein said LipDH is in direct equilibrium with theNAD⁺/NADH pool in the mitochondria.
 12. The method of claim 1, whereinthe stimulus causes both the tissue subject to evaluation and thecorresponding control tissue to change their respective steady-statefluorescence anisotropies (A) due to changes in the rotationalcorrelation time (φ) of any segment of LipDH free to rotate by amagnitude that exceeds the stimulus induced changes in the fluorescencelifetimes (τ) of both tissues according to the following equation:$\frac{A_{o}}{\overset{\_}{A}} = {{1 + \frac{\tau}{\phi}} = {1 + \sigma}}$where A₀ is a limiting value in the absence of rotation given by therelative orientation of the absorption and emission dipole transitionmoments of LipDH and σ is the ratio of τ/φ.